CHAPTER ADHESION PATHOLOGY 5 Jeffrey E.Saffitz

Introduction vested with a continuous which provides a considerable degree of electrical insulation. Elec- trical activation of the heart requires intercellular Significant progress has been made in identifying sin- transfer of current, a process that can only occur at gle gene mutations responsible for causing human car- gap junctions [2]. Thus, the number, size, and distri- diomyopathies. Although many of these monogenic cardiomyopathies are rare, insights into pathogenesis bution of gap junctions are important determinants by identification of the responsible mutations can of impulse propagation in . Further- provide clues about mechanisms in more common more, alterations in the structure or function of gap forms of heart disease. We have studied a group of hu- junctions can give rise to conduction disturbances man cardiomyopathies caused by mutations in genes that may contribute to arrhythmogenesis. encoding proteins that function as linkers in cell-cell There is an intimate structural, functional, and adhesion junctions. These heart diseases, which we spatial relationship between gap junctions and me- have termed cell-cell junction cardiomyopathies,are chanical junctions in cardiac myocytes. Cardiac my- caused by mutations in intracellular proteins that link ocytes are connected by extremely large intercellular adhesion molecules at adherens junctions and desmo- junctions, which, presumably, have evolved to sub- somes to the myocyte . Among the genes serve the specialized electrical and contractile func- implicated in these diseases are those encoding desmo- tion of these cells. For example, because cardiac my- , , and plakophilin-2. These muta- ocytes contract, they require more extensive and ro- tions have both dominant and recessive patterns of in- bust adhesion junctions than noncontractile cells in heritance and are associated with clinical phenotypes other solid organs. It is no surprise, therefore, that ad- of arrhythmogenic right ventricular cardiomyopathy/ herens junctions and , organelles re- dysplasia (ARVC/D) or dilat cardiomyopathy (DCM), sponsible for physically connecting one cardiac my- with or without hair and abnormalities [1]. Com- ocyte to another, are highly concentrated at the ends mon features of the cell-cell junction cardiomy- of individual cells where they form elaborate com- opathies are a high incidence of syncope, ventricular plexes that can be readily identified at the light mi- arrhythmias, and sudden cardiac death. This observa- croscopic level of resolution and which have been giv- tion suggests that alterations in intercellular adhesion en a specific name – the intercalated disk. Intercalat- caused by defects in cell-cell mechanical junctions may ed disks are composed mainly of arrays of adherens create anatomic substrates that are particularly con- junctions, each located at the end of a row of sar- ducive to the development of lethal ventricular ar- comeres. These junctions act as bridges which link rhythmias. Our work in this area has focused on the filaments within sarcomeres of neighboring hypothesis that defective mechanical linkage in the cells. Interspersed among the adherens junctions are cell-cell junction cardiomyopathies causes remodeling desmosomes, which also provide mechanical coupling of gap junctions, which, in turn, can give rise to con- and link cell- junctions to fila- duction abnormalities that may contribute to the high ments of the cytoskeleton. Because electrical activa- incidence of sudden death in these patients. tion of the heart requires intercellular transfer of cur- rent, cardiac myocytes also have a special requirement for extensive electrical coupling. Indeed, gap junctions Electrical Coupling at Gap Junctions in the Heart interconnecting ventricular myocytes are among the largest in living systems, presumably reflecting a need Cardiac muscle is not a true electrical syncytium. for many low-resistance electrical communicating Rather, it is composed of individual cells, each in- channels between cells to ensure safe conduction. 46 Jeffrey E. Saffitz

However, membrane regions containing gap junc- cell-cell junctions and the cardiac myocyte cytoskele- tions are rigid and nonfluid because of the high con- ton. Such defective mechanical linkage may lead to di- centration of protein within the lipid bilayer, and as minished force transmission which, in turn, can lead a result, these regions are vulnerable to shear stress. It to myocyte injury, tissue remodeling, and a clinical is no surprise, therefore, that gap junctions in cardiac picture characterized by contractile dysfunction and myocytes are located at intercalated disks where they cardiomyopathy. In addition, abnormal mechanical are virtually surrounded by mechanical junctions. linkage can destabilize the sarcolemmas of adjacent Presumably, these mechanical junctions act like “spot cells. This may lead to remodeling which welds” to create membrane domains that are protect- could contribute to slow conduction, arrhythmias, ed from shear stress caused by contractile activities of and sudden death in patients with cell-cell junction neighboring cells and which facilitate assembly and cardiomyopathies. As briefly presented below, we have maintenance of large arrays of intercellular electrical tested this hypothesis through multiple approaches. channels. On the basis of these and related observa- First, we have characterized the distribution of inter- tions, we have proposed that the extent to which car- calated disk proteins in cardiac tissues from patients diac myocytes can become electrically coupled de- and have shown that, as a general rule, the cell-cell pends on their degree of mechanical coupling [3]. As junction cardiomyopathies are associated with re- shown in Fig. 5.1, this hypothesis provides a mecha- modeling of gap junctions. Second, we have identified nistic link between contractile dysfunction and elec- electrophysiological phenotypes in genetically engi- trical dysfunction in the cell-cell junction cardiomy- neered mouse models of the human cardiomy- opathies. A genetic defect in a protein in cell-cell ad- opathies. Third, we have elucidated signaling path- hesion junctions may lead to unstable mechanical ways that coordinately regulate expression of me- linkage between cells and/or discontinuities between chanical and electrical junction proteins in cardiac myocytes in response to mechanical stress.

Linking Electrical and Contractile Dysfunction in the Altered Expression and Distribution of Cell-Cell Cell-Cell Junction Cardiomyopathies Junction Proteins in Human Cardiomyopathies

To test the hypothesis that defects in the adhesion Genetic Defect in a Protein in Cell-Cell Adhesion Junctions junction-cytoskeleton network disrupt gap junctions, we analyzed ventricular tissues from patients with Naxos disease, a cardiocutaneous syndrome consist- ing of the clinical triad of woolly hair, palmoplantar Abnormal Mechanical Linkage keratoderma, and ARVC/D [4]. Patients with Naxos at Cell-Cell Junctions and/or disease are readily identified early in life because of Discontinuities Between Junctions the distinctive cutaneous features (the disease is and the Cytoskeleton 100% penetrant) [5, 6]. Naxos disease is associated with a particularly high incidence of sudden cardiac death. Approximately 60% of affected children pre- Destabilization sent with syncope and/or aborted sudden death, and Diminished Force the annual risk of arrhythmic death is 2.3% [6]. Nax- Transmission of Sarcolemmas of Adjacent Cells os disease is caused by a recessive mutation in the gene encoding plakoglobin [7]. The mutation is a deletion of nucleotides 2157 and 2158 which causes Myocyte Injury Gap Junction a frame-shift resulting in premature termination and Myocardial Remodeling Remodeling expression of a truncated protein lacking 56 residues at the C-terminus [7]. We characterized the distrib- Electrical Dysfunction ution of cell-cell junction proteins in fixed ventricu- Contractile Dysfunction Slow Conduction lar tissues from autopsy of Naxos disease patients us- Cardiomyopathy Arrhythmias ing confocal immunofluorescence microscopy, and Sudden Death also performed immunoblotting analysis on frozen, Fig. 5.1 • A flow chart illustrating a proposed mechanism unfixed cardiac tissue from an affected child who linking electrical and contractile dysfunction in the cell-cell died of acute leukemia before overt clinical or patho- junction cardiomyopathies logical evidence of ARVC/D had developed. Im- CHAPTER 5 • Cell Adhesion Pathology 47

Fig. 5.2 • Representative confocal microscopy images of left ventricle from control and Naxos disease stained with specific antibodies against selected intercellular junction proteins munoreactive signal for plakoglobin, the mutant insult” such as the pathological changes in the right protein in Naxos disease, was dramatically reduced at ventricle in ARVC/D. intercalated disks in both ventricles from all Naxos We have also characterized the distribution of disease patients whereas signals for N-, cell-cell junction proteins in Carvajal syndrome, a car- and desmocollin-2 appeared to be nor- diocutaneous syndrome characterized by woolly hair, mal (Fig. 5.2) [4]. There was also a striking reduction palmoplantar keratoderma, and a diffuse cardiomy- in the amount of junctional signal for connexin43 opathy that is distinct from ARVC/D [8]. Complex (C×43), the major ventricular gap junction protein, ventricular arrhythmias and conduction disturbances in both the right and left ventricles in Naxos disease, are prominent in Carvajal syndrome. Affected chil- including the individual who died before ARVC/D dren usually die before the age of 20, apparently due had become manifest clinically or pathologically to both pump dysfunction and lethal arrhythmias [8]. (Fig. 5.2) [4]. Electron microscopy revealed smaller Carvajal syndrome is caused by a recessive single nu- and fewer gap junctions interconnecting ventricular cleotide deletion mutation in desmoplakin leading to myocytes, providing independent evidence of gap a premature stop-codon and truncation of the C-ter- junction remodeling. Immunoblotting revealed that minal desmin-binding domain [9]. We described the truncated plakoglobin was expressed abundantly in pathology of Carvajal syndrome and analyzed the dis- the myocardium even though it failed to localize nor- tribution of cell-cell junction proteins in the heart of mally at intercellular junctions. Similarly, although an 11-year-old girl from Ecuador [10]. The heart was C×43 signal at gap junctions was dramatically re- markedly enlarged. The left ventricle was widely di- duced, total C×43 protein content assessed by im- lated and showed shallow posterior and antero-sep- munoblotting showed little or no reduction. How- tal aneurysms with mural thrombosis (Fig. 5.3). The ever, the highly phosphorylated P2-isoform of C×43, right ventricle also showed discrete aneurysms in- which is selectively located in the junctional pool, was volving inferior, atypical, and infundibular regions missing [4]. These observations suggest that remod- (Fig. 5.3). Interestingly, these same right ventricular eling of gap junctions in Naxos disease is not related areas also show the greatest abnormalities in ARVC/D to changes in C×43 expression per se, but rather to an (the so-called triangle of dysplasia), but there was no inability to assemble and/or maintain large gap junc- gross or microscopic evidence of fatty replacement of tion channel arrays. The degree of gap junction re- right or left ventricular muscle in Carvajal Syndrome. modeling observed in Naxos disease patients is suf- Confocal microscopy revealed that immunoreactive ficient to cause conduction slowing, which could signals for both desmoplakin (the mutant protein) contribute to the characteristic widening of the QRS and plakoglobin were markedly diminished at inter- complex in the right precordial leads. Although un- calated disks, presumably reflecting altered interac- coupling at gap junctions may not, by itself, cause ar- tions between these two binding partners [10]. The rhythmias, it could produce a substrate that pro- protein desmin was distributed motes arrhythmias when combined with a “second in a normal sarcomeric pattern but it failed to local- 48 Jeffrey E. Saffitz

ab c

d e

Fig. 5.3 • Gross photographs of the right (a, b) and left (c-e) sides of the heart in Carvajal syndrome. Both ventricles con- tained discrete regions of aneurysmal wall thinning.Affected areas included the subtricuspid posterior right ventricle (tran- silluminated area in b), the posterior basal portion of the left ventricle (area marked by asterisk in d), and the antero-sep- tal portion of the left ventricle which was lined by a mural thrombus (e) ize properly at intercalated disks. This indicates that The first mouse line we characterized was a mod- interactions between desmin and desmoplakin are el of human desmin-related cardiomyopathy created disrupted in Carvajal Syndrome, which, according to by X.J. Wang in the laboratory of Jeffrey Robbins our hypothesis, would cause remodeling of gap junc- [11]. We were attracted to this model because we an- tions. As predicted, C×43 signal at junctions was ticipated that it might exhibit altered desmin-desmo- markedly diminished. These results provide further some interactions and, therefore, recapitulate Carva- evidence that abnormal protein-protein interactions jal syndrome. Human desmin-related skeletal and at intercellular junctions cause both contractile and cardiomyopathies have been attributed to a 7-amino electrical dysfunction in Carvajal Syndrome. acid deletion mutation (R173-E179) and several mis- Taken together, our studies of human cell-cell sense mutations in desmin. To determine whether the junction cardiomyopathies have led to two major R173-E179 deletion was sufficient to cause desmin- conclusions: (1) remodeling of gap junctions is a con- related cardiomyopathy, Wang et al. [11] produced sistent and prominent feature of the cell-cell junction transgenic mice with cardiac-specific expression of cardiomyopathies, and (2) specific patterns of ab- the 7-amino acid deletion mutation in desmin (D7- normal localization of mechanical junction proteins des) implicated in the human disease. In their initial at intercalated disks correlate with cardiomyopathy description of this model, they showed that D7-des disease phenotypes. mice exhibit features of human desmin-related car- diomyopathy including intracellular accumulation of desmin, disruption of the desmin filament network, Mouse Models of Human Cardiomyopathies misalignment of myofibrils, and diminished respon- siveness to α-adrenergic agonist stimulation [11]. Delineation of structural and molecular pathology in To test the hypothesis that expression of D7-des human tissues is essential to understanding the cell- disrupts the linkage between desmosomes and the cy- cell junction cardiomyopathies; yet to elucidate mech- toskeleton and leads to remodeling of gap junctions, anisms of disease, we have turned to analysis of mouse we characterized the expression and localization of in- models. tercellular junction proteins and searched for an elec- CHAPTER 5 • Cell Adhesion Pathology 49

Fig. 5.4 • Representative confocal immunofluorescence images showing the amount of C×43 immunoreactive signal at cell-cell junctions in left ventricular myocardium from a nontransgenic control mouse (Con),a transgenic mouse expressing wild-ype desmin (WT-des), and a transgenic mouse expressing D7-des trophysiological phenotype [12]. As predicted by stud- tricular myocytes are grown on silicone membranes ies of the human cell-cell junction cardiomyopathies, and subjected to uniaxial pulsatile stretch [13]. Im- C×43 signal at intercalated disks was decreased by ap- position of this mechanical load rapidly induces a hy- proximately threefold in D7-des hearts due to signifi- pertrophic response which can be rigorously quanti- cant reductions in both the number and mean size of fied and characterized. An important feature of this individual gap junctions (Fig. 5.4). The amount of im- response is a marked increase in C×43 expression and munoreactive signal at intercalated disks was also re- enhanced intercellular coupling. After only 1h of duced significantly for selected adhesion molecules stretch (110% of resting length at 3 Hz), expression and linker proteins of both desmosomes and adherens of C×43 is increased by approximately twofold, re- junctions, and desmin-desmosomal interactions were sulting in a significant increase in both the number completely disrupted [12]. Quantitative electron mi- of gap junctions and the velocity of impulse propa- croscopy showed decreased gap junction density in gation [13]. We have shown previously that upregu- D7-des mice, providing independent evidence of gap lation of C×43 expression is mediated by stretch-in- junction remodeling, but immunoblotting showed no duced secretion of vascular endothelial growth fac- reduction in the total tissue content of C×43 and me- tor (VEGF) which acts in an autocrine fashion [14]. chanical junction proteins. These observations are Incubation of cells with exogenous VEGF for 1h in- consistent with findings in Naxos disease, suggesting creases C×43 expression by an amount roughly equal that diminished localization of cell-cell junction pro- to that seen after 1h of pulsatile stretch. Moreover, teins at intercalated disks is not due to insufficient pro- stretch-induced upregulation of C×43 expression tein expression but, rather, to failure of these proteins can be blocked by stretching cells in the presence of to assemble properly within electrical and mechanical anti-VEGF or anti-VEGF receptor antibodies. junctions. We also showed, using optical mapping, that To determine whether stretch-induced formation remodeling of gap junctions in D7-des mice slows ven- of new gap junctions requires concomitant assembly tricular conduction [12]. These results indicate, there- of new mechanical junctions, we measured changes in fore, that a defect in a protein conventionally thought mechanical junction protein expression in cells sub- to fulfill a strictly mechanical function in the heart can jected to stretch [15]. The amounts of plakoglobin, also lead to electrophysiological alterations that may desmoplakin, and N-cadherin at cell-cell junctions all contribute to arrhythmogenesis. increased by at least twofold in myocytes subjected to 1h of pulsatile stretch (Fig. 5.5). However, VEGF se- cretion plays no role in this process. For example, ad- Mechanisms Regulating Expression of Cell-Cell dition of exogenous VEGF does not affect plakoglo- Junction Proteins in Response to Mechanical bin, desmoplakin, or N-cadherin expression, nor is Load stretch-induced upregulation of these proteins blocked by anti-VEGF antibodies. To further define To elucidate mechanisms regulating expression of in- the responsible mechanisms, we studied the role of fo- tercellular junction proteins, we developed an in vit- cal adhesion kinase (FAK), which is phosphorylated ro system in which monolayers of neonatal rat ven- in response to engagement and activates 50 Jeffrey E. Saffitz a Fig.5.5 • Representative confo- cal immunofluorescence im- ages (a) (top = control,bottom = stretch and quantitative confo- cal microscopy data (b) show- ing the effects of stretch on expression of C×43 and the mechanical junction proteins, plakoglobin, desmoplakin, and N-cadherin.* p<0.05 compared with control

12 Control 10 * Stretch

8 *

6 * 4 *

2

0

b Cx43 Plakoglobin Desm oplakin N-cadherin

multiple intracellular signaling molecules including by disparate mechanisms. C×43 expression is regu- src kinase. We infected cardiac myocytes with an ade- lated by autocrine actions of chemical mediators se- novirus containing GFP-FRNK, a GFP-tagged domi- creted during stretch, whereas adhesion junction pro- nant-negative inhibitor of FAK-dependant signaling, teins are regulated by intracellular mechanotrans- and then subjected cells to pulsatile stretch [15]. duction pathways initiated via FAK and dependent on FRNK blocked stretch-induced upregulation of both downstream activation of src kinase. electrical (C×43) and mechanical (N-cadherin, desmoplakin, and plakoglobin) junction proteins. In- fection of cells with virus expressing GFP alone had Conclusions no effect. Addition of exogenous VEGF to FRNK-in- fected cells upregulated expression of C×43 but not We have proposed a unified hypothesis that links con- mechanical junction proteins. Conditioned medium tractile and electrical dysfunction in the cell-cell junc- removed from uninfected cells after 1h of stretch in- tion cardiomyopathies. It is based on the premise that creased C×43 expression when added to nonstretched the extent to which cardiac myocytes are coupled me- cells, and this effect was blocked by anti-VEGF anti- chanically at cell-cell adhesion junctions is a key de- bodies, but stretch-conditioned medium from FRNK- terminant of the extent to which they can be coupled infected cells had no effect on C×43 expression. Thus, electrically at gap junctions. Our observations in sev- secretion of VEGF in response to stretch requires ac- eral human cardiomyopathies indicate that genetic de- tivation of FAK. Finally, the src kinase inhibitor PP2 fects in linker proteins such as desmoplakin and plako- blocked stretch-induced upregulation of mechanical globin can create anatomic substrates of sudden death junction proteins but not C×43 [15]. These results in- by remodeling gap junctions. Molecular mechanisms dicate that mechanical load regulates expression of responsible for gap junction remodeling in the cell-cell both electrical and mechanical junctions proteins, but junction cardiomyopathies are unknown. One possi- CHAPTER 5 • Cell Adhesion Pathology 51 bility is that rates of C×43 synthesis and degradation 4. Kaplan SR, Gard JJ, Protonotarios N et al (2004) Re- are unaffected but molecules are unable to modeling of myocyte gap junctions in arrhythmogenic assemble properly in gap junctions. It must also be right ventricular cardiomyopathy due to a deletion in considered, however, that C×43 gene expression may plakoglobin (Naxos disease). Heart Rhythm 1:3-11 be altered in the cell-cell junction cardiomyopathies. 5. Protonotarios N, Tsatsopoulou AA, Gatzoulis KA (2002) Arrhythmogenic right ventricular cardiomy- Plakoglobin and other members of the fami- opathy caused by a deletion in plakoglobin (Naxos dis- ly fulfill both structural and nuclear signaling roles ease). Card Electrophysiol Rev 6:72-80 [16]. Disease-related mutations may shift the relative 6. Protonotarios N, Tsatsopoulou A, Anastasakis A et al proportions of these proteins within junctional and (2001) Genotype-phenotype assessment in autosomal cytosolic pools, which, in turn, could affect nuclear sig- recessive arrhythmogenic right ventricular cardiomy- naling mediated by plakoglobin, or β-catenin or oth- opathy (Naxos disease) caused by a deletion in plako- er related proteins. If, for example, β-catenin substi- globin. J Am Coll Cardiol 38:1477-1484 tutes for mutant plakoglobin within cell-cell junctions, 7. McKoy G, Protonotarios N, Crosby A et al (2000) then the resultant decrease in the cytosolic pool of Identification of a deletion in plakoglobin in arrhyth- β-catenin could lead to diminished expression of C×43 mogenic right ventricular cardiomyopathy with pal- and other proteins under the control of β-catenin sig- moplantar keratoderma and woolly hair (Naxos dis- ease). Lancet 355:2119-2124 naling. Thus, it is possible and perhaps even likely that 8. Carvajal-Huerta L (1998) Epidermolytic palmoplantar both altered mechanical integrity and altered nuclear keratoderma with woolly hair and dilated cardiomy- signaling underlie the pathogenesis of contractile and opathy. J Am Acad Dermatol 39:418-421 electrical dysfunction in heart muscle diseases caused 9. Norgett EE, Hatsell SJ, Carvajal-Huerta L et al (2000) by mutations in cell-cell junction proteins. Recessive mutation in desmoplakin disrupts desmo- plakin-intermediate filament interactions and causes dilated cardiomyopathy, woolly hair and keratoderma. Acknowledgements Hum Mol Genet 9:2671-2766 10. Kaplan SR, Gard JJ, Carvajal-Huerta L et al (2004) Work in the author’s laboratory was supported by Structural and molecular pathology of the heart in grants from the National Institutes of Health, March Carvajal syndrome. Cardiovasc Pathol 13:26-32 11. Wang X, Osinska H, Dorn GW 2nd et al (2001) Mouse of Dimes, American Heart Association, and the model of desmin-related cardiomyopathy. Circulation Sarnoff Endowment. 103:2402-2407 12. Gard JJ, Yamada K, Green KG et al (2005) Remodeling of gap junctions and slow conduction in a mouse References model of desmin-related cardiomyopathy. Cardiovasc Res 67:539-547 1. Protonotarios N, Tsatsopoulou A (2004) Naxos disease 13. Zhuang J, Yamada KA, Saffitz JE et al (2000) Pulsatile and Carvajal syndrome: cardiocutaneous disorders stretch remodels cell-to-cell communication in cul- that highlight the pathogenesis and broaden the spec- tured myocytes. Circ Res 87:316-322 trum of arrhythmogenic right ventricular cardiomy- 14. Pimentel RC, Yamada KA, Kléber AG et al (2002) Au- opathy. Cardiovasc Pathol 13:185-194 tocrine regulation of C×43 expression by VEGF. Circ 2. Saffitz JE, Lerner DL, Yamada KA (2004) Gap junction Res 90:671-677 distribution and regulation in the heart. In: Zipes DP, 15. Yamada K,Green KG, Samarel AM et al (2005) Distinct Jalife J, (eds) Cardiac electrophysiology: From cell to pathways regulate expression of cardiac electrical and bedside, 4th edn. Saunders, Philadelphia pp 181-191 mechanical junctions proteins in response to stretch. 3. Saffitz JE (2005) Dependence of electrical coupling on Circ Res 97:346-353 mechanical coupling in cardiac myocytes: insights 16. Conacci-Sorrell M, Zhurinsky J, Ben-Ze’ev A (2002) gained from cardiomyopathies caused by defects in The cadherin-catenin adhesion system in signaling cell-cell connections. Ann N Y Acad Sci 1047:336-344 and cancer. J Clin Invest 109:987-991